Transferrin (Tf), the iron carrier protein of the circulation, binds to specific receptors on the surfaces of most cells. The receptor/Tf complex is then internalized and, having lost its iron intracellularly, the apoTf/receptor complex recycles to the cell surface. Very little is known about how the recycling process is controlled. When human erythroleukemic K562 cells are incubated with hemin, phorbol esters or trifluoperazine (TFP), the number of Tf receptors on the cell surface is sharply reduced. In the case of TFP-induced internalization at least, the process is reversible and the site of intracellular sequestration of the receptors has been identified as juxtanuclear multivesicular bodies. After all three receptor internalizing treatments, the total complement of cellular Tf receptors is hyperphosphorylated. It is thus possible that Tf receptor phosphorylation is involved in the control of receptor recycling and internal sequestration. It is proposed, using electron microscopic cytochemistry, to determine the routes taken by Tf and its receptor in cells treated with a variety of receptor internalizing agents and also to identify the sites of internal accumulation. The involvement of protein kinase C in this process, which is suggested by the effects of phorbol esters and TFP, will be studied. Using immune precipitation of 32P-labeled Tf receptors and peptides analysis by electrophoresis/chromatography, the pattern of receptor phosphorylation after incubation of cells with receptor-internalizing agents will be compared. The sites that become labeled in each case will be localized to specific amino acids within the primary sequence of the receptor and the kinetics of phosphorylation of the various sites will be compared with those of receptor internalization to determine whether one could be the cause of the other. We have gained preliminary evidence that a protein that could be the kinase forms an intermolecular complex with the receptor and the identification of this protein will be pursued using affinity labeling and peptide mapping procedures. If it seems likely that receptor phosphorylation is indeed involved in the control of receptor recycling, K562 cell mutants will be raised that are deficient in Tf receptor internalization and intracellular processing in order to determine whether the mutant phenotype may result from incorrect receptor phosphorylation.